DESCRIPTION: This proposal, requesting 5 year support to study the physiologic function of the transcription factor NF-E2 in the process of stem cell commitment and differentiation. The Investigator proposes to use gene targeted embryonic stem cells as the primary experimental method for functional dissection. The transcription factor NFE-2 was recently purified, partially characterized, and cloned in the Principal Investigator's laboratory. It appears to be a critical regulator of gene transcription during erythroid differentiation; it may also be important during mast cell and platelet differentiation. Work by this group has suggested that NFE-2 binds to critical target sequences within the core enhancer element of the locus control region, a sequence whose "activation" seems to be necessary for expression of many genes critical to the phenotype of the mature red cell. Many, but not all, aspects of NFE-2 expression are similar to those observed with another transcription factor, GATA-1 which had been cloned and extensively characterized in this laboratory. The Investigator hypothesizes that constitutive expression of these factors occurs in all lines, but that lineage commitment and differentiation may be associated with selective loss of the factors from some lineages, coupled with persistent or increased expression in others. NFE-2 is a heterodymer consisting of a 45 kD unique subunit and an 18 kD subunit that is widely expressed. This proposal focuses upon the 45 kD subunit, which will be referred to as NFE-2. NFE-2 is a basic region-leucine zipper (B-zip) transcription factor, which bears homology to several other factors known to be important for regulation in other systems. Its potential importance in hematopoiesis is indicated by the fact that a subtle mutation produces the mk mouse which suffers from a microcytic hypochromic anemia with intrinsic defects in both blood cell production and the intestine. Interestingly, NFE-2 is expressed inductively in the intestine of severely anemic mice, suggesting that it may play a role in iron transport. There is thus ample evidence that this protein subunit of the NFE-2 transcription factor plays a critical role in blood cell differentiation. The First Specific Aim is focused upon construction of chimeric mice carrying ES cells with a single disruption of the NF-E2 gene. Heterozygous and homozygous animals deficient in the gene will be generated by breeding, and their phenotype characterized by hematologic and pathologic examination of embryos and/or adult tissues. The Second Aim will utilize a similar approach to generate chimeric mice, in whom defects in hematopoiesis can be isolated from defects, particularly lethal defects, in other organ systems. Isoenzyme markers and lacZ reporter genes will be used to track new clones and colonies bearing the gene disrupted cells derived from the ES cell lines.In vitro colony assays from the chimeras will be utilized to assess the ability of the ES cell-derived progenitors to generate erythroid, mass cell, or megakaryocytic lineages.The Third Aim will examine blood cell differentiation directly in embryoid body (EB) of the disrupted ES cells, while the Fourth Aim will attempt to develop an MELC line with disruption of the NFE-2 locus. The relative ease of use and high quantities of MELC will permit development of assays for DNA's hypersensitivity, transcription of genes regulated by NFE-2, development of substracted cDNA libraries, etc. The Fifth Specific Aim will utilize normal and mutated forms of NFE-2 genes to rescue the phenotypes documented in the other specific aims. The proposal contains much preliminary data directed toward each of the Specific Aims. The necessary cDNA reagents have been cloned, and most of the technology proposed in the application has been utilized by the Applicant for a study of the GATA-1 gene.